Estrogenic compounds in combination with progestogenic compounds in hormone-replacement therapy

ABSTRACT

One aspect of the present invention relates to a method of hormone replacement in mammals, which method comprises the oral administration of an estrogenic component and a progestogenic component to a mammal in an effective amount to prevent or treat symptoms of hypoestrogenism, wherein the estrogenic component is selected from the group consisting of substances represented by the above formula in which formula R 1 , R 2 , R 3 , R 4  independently are a hydrogen atom, a hydroxyl group or an alkoxy group with 1-5 carbon atoms; each of R 5 , R 6 , R 7  is a hydroxyl group; and no more than 3 of R 1 , R 2 , R 3 , R 4  are hydrogen atoms; precursors capable of liberating a substance according to the aforementioned formula when used in the present method; and mixtures of one or more of the aforementioned substances and/or precursors. Another aspect of the invention concerns a pharmaceutical kit comprising oral dosage units that contain the aforementioned estrogenic component and a progestogenic component as well as an androgenic component.

TECHNICAL FIELD OF THE INVENTION

The present invention relates to a method of hormone replacement inmammals. More particularly the invention is concerned with a method ofhormone replacement that comprises the oral administration to a mammalof a combination of an estrogenic component and a progestogeniccomponent in an effective amount to prevent or treat symptoms ofhypoestrogenism.

BACKGROUND OF THE INVENTION

In hormone replacement therapy (HRT), sometimes also referred to asestrogen replacement therapy, estrogens are administered to prevent ortreat symptoms resulting from estrogen deficiency or hypoestrogenism.Hypoestrogenism can occur in both females and males, and can lead todisorders and ailments such as osteoporosis (loss of bone mass),arteriosclerosis, climacteric symptoms such as hot flushes (flashes),sweats, urogenital atrophy, mood disturbances, insomnia, palpitations.Estrogen deficiency has also been associated with cognitive disturbancesand Alzheimer's disease.

Hypoestrogenism, and in particular chronic hypoestrogenism, isfrequently observed in (peri-)menopausal and post-menopausal women.However, it can also result from hypogonadism or castration, as well asfrom primary ovarian failure, treatment of e.g. breast cancer witharomatase inhibitor and gonadotropin-releasing hormone analoguetreatment of benign gynaecological diseases such as endometriosis,adenomyosis, uterine fibroids (leiomyomas), dysmenorrhoea, menorrhagiaand metrorrhagia.

HRT employs continuous administration of effective amounts of anestrogen for prolonged periods of time. The administration of estrogenshas been associated, however, with endometrial proliferation in womenand it is now widely accepted that “unopposed” estrogen therapysubstantially increases the risk of endometrial cancer (Cushing et al.,1998. Obstet. Gynecol. 91, 35-39; Tavani et al., 1999. Drugs Aging, 14,347-357). There is also evidence of a significant increase in breastcancer with long-term (10-15 years) use of estrogen therapy (Tavani etal., 1999. Drugs Aging, 14, 347-357; Pike et al., 2000. Steroids, 65,659-664).

In order to counteract the negative effects of unopposed estrogentherapy, adjunctive progestogen treatment is nowadays commonly applied.Regular progestogen administration is believed to inhibit the continualestrogen stimulation of the endometrium through an anti-proliferativeeffect and appears to reduce the incidence of endometrial carcinoma inpost-menopausal women receiving estrogen replacement therapy (Beral etal., 1999. J. Epidemiol. Biostat., 4, 191-210). Such an adjunctivetreatment, generally using synthetic progestogens, is given either incontinuous combined regimens with estrogen, or added sequentially,typically for about 14 days each month, to continuous estrogentreatment.

Endogenous and exogenous estrogens fulfil important central nervous andmetabolic functions in the female organism: normal estrogen levels makea decisive contribution to a woman's well-being. Notwithstanding thewidespread use of estrogens in HRT methods, there are still someunsolved problems. Known estrogens, in particular the biogenic estrogens(i.e. estrogens naturally occurring in the human body), show seriouspharmacokinetic deficits. Biogenic estrogens such as estradiol, estrone,estrone sulphate, esters of estradiol and estriol become bioavailableonly to a very low degree when taken orally. This degree may vary somuch from person to person that general dosage recommendations cannot begiven. Fast elimination of these estrogens from the blood is anotherrelated problem. For instance, for the main human biogenic estrogen17β-estradiol the half-life is around 1 hour. As a result, betweenseparate (daily) administration events, blood serum levels of suchbiogenic estrogens tend to fluctuate considerably. Thus, shortly afteradministration the serum concentration is usually several times higherthan the optimum concentration. In addition, if the next administrationevent is delayed, serum concentrations will quickly decrease to a levelwhere the estrogen is no longer physiologically active.

The most important synthetically altered estrogenic steroid is17α-ethinyl estradiol (EE). This estrogen is hardly used in HRT methodsbecause prolonged administration of EE has been associated with anincreased risk of thromboembolism, which is deemed to be particularlydetrimental in menopausal and post-menopausal females. Apart from EE,mestranol has been used in a few cases; mestranol is a “prodrug” that ismetabolised to EE in the organism. When applied orally to humans, EE hasa much better bioavailability than the biogenic estrogens mentionedabove, but its oral bioavailability varies to a large extent fromindividual to individual. Several authors have pointed to this as wellas to the fact that concentrations in the blood proved to be highlyfluctuating after oral application of this substance.

In addition to pharmacokinetic problems, the known estrogens also showpharmacodynamic deficits. After resorption from the intestinal lumen,orally applied active ingredients enter the organism via the liver. Thisfact is of specific importance for estrogenic agents as the liver is atarget organ for estrogens; oral intake of estrogens results in strongestrogenic effects in the liver. The secretion activity that iscontrolled by estrogens in the human liver includes increased synthesisof transport proteins CBG, SHBG, TBG, several factors that are importantfor the physiology of blood clotting, and lipoproteins. If biogenicestrogens are introduced to the female organism while avoiding passagethrough the liver (e.g. by transdermal application), the liver functionsmentioned remain largely unchanged. Therapeutically equivalent doses ofbiogenic estrogens, when applied orally, result in clear responses ofhepatic parameters, such as increase of SHBG, CBG, angiotensinogen andHDL (high density lipoprotein). These hepatic effects of estrogens arealso observed when equine estrogen formulations (so-called conjugatedestrogens) are used. Ethinyl estradiol and diethylstilbestrol (DES) havean even greater hepatic estrogenicity. Elger et al., J. Steroid Biochem.Molec. Biol. (1995), 55(3/4), 395-403, have reported that EE or DES havemuch higher hepato-cellular than systemic estrogenicity: in relation toFSH-secretion inhibitory activity these estrogens are 4-18 times moreactive in the liver than estrone sulfate.

The aforementioned deficits are of considerable clinical significancewhen commonly known biogenic and synthetic estrogens are applied.Consequently, there is an as yet unmet need for estrogens that do notdisplay these deficits and which can suitably be administered orally inHRT methods to effectively replace endogenous ovarian secretion ofestradiol, i.e. to treat or prevent symptoms of hypoestrogenism.

SUMMARY OF THE INVENTION

The inventors have surprisingly found that these objectives are met byestrogenic substances that are represented by the following formula

in which formula R₁, R₂, R₃, R₄ independently are a hydrogen atom, ahydroxyl group or an alkoxy group with 1-5 carbon atoms; each of R₅, R₆,R₇ is a hydroxyl group; and no more than 3 of R₁, R₂, R₃, R₄ arehydrogen atoms.

A known representative of this group of estrogenic substances is1,3,5(10)-estratrien-3,15α,16α,17β-tetrol, also known by the names ofestetrol, oestetrol and 15α-hydroxyestriol. Estetrol is an estrogen thatis produced by the fetal liver during human pregnancy. Unconjugatedestetrol levels in maternal plasma peak at about 1.2 ng/ml at termpregnancy and are about 12 times higher in fetal than in maternal plasma(Tulchinsky et al., 1975. J. Clin. Endocrinol. Metab., 40, 560-567).

In 1970, Fishman et al., “Fate of 15α-hydroxyestriol-³H in Adult Man”, JClin Endocrinol Metab (1970) 31, 436-438, reported the results of astudy wherein tritium labeled 15α-hydroxyestriol (estetrol) wasadministered intravenously to two adult women. It was found that theestetrol was rapidly and completely excreted in urine as theglucosiduronate and that virtually no metabolism except for conjugationtook place.

Between 1975 and 1985 several researchers have investigated theproperties of estetrol and reported on its estrogenic potency anduterotrophic activity. The most relevant publications that were issuedduring this period are mentioned below:

-   -   Levine et al., 1984. Uterine vascular effects of estetrol in        nonpregnant ewes. Am. J. Obstet. Gynecol., 148:73, 735-738:        “When intravenously administered in nonpregnant ewes, estetrol        is 15 to 30 times less potent than estriol and 17β-estradiol in        uterine vasodilation”.    -   Jozan et al., 1981. Different effects of oestradiol, oestriol,        oestetrol and of oestrone on human breast cancer cells (MCF-7)        in long term tissue culture. Acta Endocrinologica, 98, 73-80:        “Estetrol agonistic potency is 2% of the magnitude observed for        17β-estradiol in in vitro cell proliferation”.    -   Holinka et al., 1980. Comparison of effects of estetrol and        tamoxifen with those of estriol and estradiol on the immature        rat uterus. Biol. Reprod. 22, 913-926: “Subcutaneously        administered estetrol has very weak uterotrophic activity and is        considerable less potent than 17β-estradiol and estriol”.    -   Holinka et al., 1979. In vivo effects of estetrol on the        immature rat uterus. Biol. Reprod. 20, 242-246: “Subcutaneously        administered estetrol has very weak uterotrophic activity and is        considerable less potent than 17(3-estradiol and estriol”.    -   Tseng et al., 1978. Heterogeneity of saturable estradiol binding        sites in nuclei of human endometrium. Estetrol studies. J.        Steroid Biochem. 9, 1145-1148: “Relative binding of estetrol to        estrogen receptors in the human endometrium is 1.5% of        17β-estradiol”.    -   Martucci et al., 1977. Direction of estradiol metabolism as a        control of its, hormonal action-uterotrophic activity of        estradiol metabolites. Endocrin. 101, 1709-1715: “Continuous        administration of estetrol, from a subcutaneous depot shows very        weak uterotrophic activity and is considerably less potent than        17β-estradiol and estriol”.    -   Tseng et al., 1976. Competition of estetrol and ethynylestradiol        with estradiol for nuclear binding in human endometrium. J.        Steroid Biochem. 7, 817-822: “The relative binding constant of        estetrol binding to the estrogen receptor in the human        endometrium is 6.25% compared to 17β-estradiol (100%)”.    -   Martucci et al., 1976. Uterine estrogen receptor binding of        catecholestrogens and of estetrol        (1,3,5(10)-estratriene-3,15alpha,16alpha,17beta-tetrol).        Steroids, 27, 325-333: “Relative binding affinity of estetrol to        rat uterine cytosol estrogen receptor is 0.5% of 17β-estradiol        (100%). Furthermore, the relative binding affinity of estetrol        to rat uterine nuclear estrogen receptor is 0.3% of        17β-estradiol (100%)”.

All of the above publications have in common that the authors haveinvestigated the estrogenic potency of estetrol. Without exception theyall conclude that estetrol is a weak estrogen. In some of the citedarticles the estrogenic potency of estetrol has been found to be lowerthan that of another biogenic estrogen, namely, 17β-estradiol, which isconsidered to be a relatively weak estrogen (e.g. compared to ethinylestradiol). With these findings in mind, it is not surprising that theinterest in estetrol has dwindled since the early eighties and that nopublications on the properties of estetrol have been issued since.

U.S. Pat. No. 5,468,736 (Hodgen) describes a method of hormonereplacement therapy involving the administration of estrogen togetherwith an amount of antiprogestin (antiprogestogen), which inhibitsestrogen-induced endometrial proliferation in women. In Example 3 thecombined use of estetrol and lilopristone is mentioned. No clues aregiven in the examples as to the mode and frequency of administration orregarding the dosage level employed. A disadvantage associated with theuse of antiprogestogens, such as lilopristone, is the risk of inducingabnormal endometrial morphology, i.e. cystic hyperplasia, as has beenobserved in women who received an antiprogestogen treatment againstendometriosis (Murphy et al., 1995. Fertil. Steril., 95, 761-766).

U.S. Pat. No. 5,340,586 (Pike et al.) is concerned with compositions andmethods which are effective to treat oophorectomised women, wherein aneffective amount of an estrogenic composition and an androgeniccomposition are provided over a period of time. In the U.S. patent it isstated that natural and synthetic estrogenic compositions that can beused include natural estrogenic hormones and congeners, including butnot limited to estradiol, estradiol benzoate, estradiol cypionate,estradiol valerate, estrone, diethylstilbestrol, piperazine estronesulfate, ethinyl estradiol, mestranol, polyestradiol phosphate, estriol,estriol hemisuccinate, quinestrol, estropipate, pinestrol and estronepotassium sulfate, and furthermore that equine estrogens, such asequilelinin, equilelinin sulfate and estetrol, may also be employed.Except for the exhaustive inventory of known estrogens, no otherreference to estetrol (which is erroneously referred to as an equineestrogen) is made in this U.S. patent.

The same exhaustive list of estrogens is found in the following patentdocuments:

-   -   U.S. Pat. No. 4,762,717 (Crowley): A contraceptive method        comprising the sequential administration of (1) a combination of        luteinizing hormone releasing hormone (LHRH) and estrogen        and (2) a combination of LHRH and estrogen and progestogen.    -   U.S. Pat. No. 5,130,137 (Crowley): A method of treating benign        ovarian secretory disorder comprising the sequential        administration of (1) a combination of luteinizing hormone        releasing hormone (LHRH) and estrogen and (2) a combination of        LHRH and estrogen and progestogen.    -   U.S. Pat. No. 5,211,952 (Spicer et al.): A contraceptive method        comprising administering a gonadotropin hormone releasing        hormone (GnRH) composition in an amount effective to inhibit        ovulation and administering estrogen and progestogen to maintain        serum levels above a defined minimum level.    -   U.S. Pat. No. 5,340,584 (Spicer et al.): A method for preventing        conception or for treating benign gynaecological disorders        comprising administering a GnRH composition for a first period        of time in an amount effective to suppress ovarian estrogen and        progesterone production, simultaneously administering an        estrogenic composition in an amount effective to prevent        symptoms of estrogen deficiency and simultaneously administering        a progestogen in an amount effective to maintain serum level of        said progestogen at a level effective to decrease endometrial        cell proliferation.    -   U.S. Pat. No. 5,340,585 (Pike et al.): A method of treating        benign gynaecological disorders in a patient in whom the risk of        endometrial stimulation by estrogenic compositions is minimised        or absent, comprising administering a GnRH composition in an        amount effective to suppress ovarian estrogen and progesterone        production and administering an estrogenic composition in an        amount effective to prevent symptoms of estrogen deficiency.    -   WO 00/73416 (Yifang et al.): A method for regulating the        fertility of a host, comprising contacting host ovarian cells        with a safe and effective amount of a pharmaceutical composition        comprising an antisense oligonucleotide that is complementary to        the nucleotide sequence of the follicle stimulating hormone        (FSH) receptor. The possibility of combined administration of        such an antisense oligonucleotide with an estrogenic steroid is        mentioned in the application.

The benefits of the present invention may be realised without theco-administration of anti-progestogens, LHRH compositions, GnRHcompositions and/or antisense oligonucleotides that are complementary tothe nucleotide sequence of the follicle stimulating hormone (FSH)receptor as proposed in the aforementioned patents. Also, the presentinvention may suitably be applied in individuals who have not beenoophorectomised, or in whom the risk of endometrial stimulation byestrogenic compositions is not minimised or absent, other than throughthe co-administration of a progestogen. Furthermore the present methoddoes not require the use of a slow release formulation as is dictated bymost of the aforementioned publications.

It is noted that none of the aforementioned publications describe theoral administration of estetrol. The only modes of administrationdescribed therein are intravenous and subcutaneous (depot)administration. For each of these modes of administration it can beconcluded that the performance of estetrol is very much inferior to thatof e.g. 17β-estradiol. Given that there was no reason to assume that adifferent outcome might be obtained in case of oral administration, itis not surprising that oral administration of estetrol has not beenpursued and that no reports to this effect can be found in the priorart.

In view of the low estrogenic potency of the estetrol-like substancesthat are employed in accordance with the invention, it is surprisingthat these substances can effectively be used in HRT methods,particularly in HRT methods that employ oral administration of suchsubstances. Although the inventors do not wish to be bound by theory, itis believed that the unexpected efficacy of orally administeredestetrol-like substances results from the combination of unforeseenfavourable pharmacokinetic (ADME) and pharmacodynamic properties ofthese substances.

As regards the pharmacokinetic properties of the present estrogenicsubstances the inventors have discovered that their oral bioavailabilityis surprisingly high and that their in vivo half-life is considerablylonger than that of other biogenic estrogens. Thus, even though estetroland estetrol-like substances have relatively low estrogenic potency,they may effectively be employed in an oral HRT method because their lowpotency is compensated for by a relatively high oral bioavailability incombination with a high metabolic stability, as demonstrated by a longhalf-life.

An important advantage of oral administration of estetrol andestetrol-like substances resides in the fact that the hepatic effects ofestetrol-like substances are deemed to be minimal since they are hardlymetabolised during the so called “first pass”. The first-pass effect ofdrugs given orally, refers to the process of drug degradation by theliver during a drug's transition from initial ingestion to circulationin the blood stream.

Another advantageous property of the present estrogenic substancesresides in the fact that sex hormone-binding globulin (SHBG) hardlybinds these estrogenic substances, meaning that, in contrast to mostknown estrogens, serum levels are representative for bio-activity andindependent of SHBG levels.

Yet another important benefit of the present estrogenic substances isderived from their relative insensitivity to interactions with otherdrugs (drug-drug interactions). It is well known that certain drugs maydecrease the effectiveness of estrogens, such as ethinyl estradiol, andother drugs may enhance their activity, resulting in possible increasedside-effects. Similarly estrogens may interfere with the metabolism ofother drugs. In general, the effect of other drugs on estrogens is dueto interference with the absorption, metabolism or excretion of theseestrogens, whereas the effect of estrogens on other drugs is due tocompetition for metabolic pathways.

The clinically most significant group of estrogen-drug interactionsoccurs with drugs that may induce hepatic microsomal enzymes which maydecrease estrogen plasma levels below therapeutic level (for example,anticonvulsant agents; phenyloin, primidone, barbiturates,carbamazepine, ethosuximide, and methosuximide; antituberculous drugssuch as rifampin; antifungal drugs such as griseofulvin). The presentestrogenic substances are less dependent on up- and downregulation ofmicrosomal liver enzymes (e.g. P450's) and also are less sensitive tocompetition with other P450 substrates. Similarly, they do not interferesignificantly in the metabolism of other drugs.

The conjugates of most estrogens, as formed in the liver, are excretedin the bile and may be broken down by gut bacteria in the colon toliberate the active hormone which can then be reabsorbed (enterohepaticrecirculation). There are clinical reports that support the view thatenterohepatic recirculation of estrogens decreases in women takingantibiotics such as ampicillin, tetracycline, etc. Conjugated forms ofthe present estrogenic substances are hardly excreted in the bile,meaning that they are substantially insensitive to drugs that doinfluence the enterohepatic recirculation of other estrogens.

The above observations serve to explain why the estrogenic substances ofthe invention hardly suffer from drug-drug interactions and thus producea very consistent, i.e. predictable, impact. Thus, the efficacy of theestrogenic substances of the invention is highly reliable.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing the total bone mineral density from theproximal tibiae of Sham- and OVX-rats orally (po) treated with17α-ethinyl estradiol (EE), estetrol (E4) or vehicle for 4 consecutiveweeks. Data are expressed as the mean values obtained for each group(n=10).

FIG. 2 is a graph showing the mean ultimate strength of the distal femurof Sham- and OVX-rats orally (po) treated with 17α-ethinyl estradiol(EE), estetrol (E4) or vehicle for 4 consecutive weeks. Data areexpressed as the mean values obtained for each group (n=10).

FIG. 3 is a graph showing the effects of estetrol (E4) and 17α-ethinylestradiol (EE) on the malonxone induced hot flush response in femaleovariectomized rats.

FIG. 4 contains graphs showing the competitive displacement of [³H]DHT(panel A) and [³H]estradiol (panel B) from the human sex hormone-bindingglobulin steroid binging site. The unlabeled steroid ligands used ascompetitors were as follows: estetrol (E4), 17α-ethinyl estradiol (EE2),17β-estradiol (E2), testosterone (T) and 5α-dihydrotestosterone (DHT).

DETAILED DESCRIPTION OF THE INVENTION

Accordingly one aspect of the present invention relates to a method ofhormone replacement in mammals, which method comprises the oraladministration of an estrogenic component and a progestogenic componentto a mammal in an effective amount to prevent or treat symptoms ofhypoestrogenism, wherein the estrogenic component is selected from thegroup consisting of:

substances represented by the following formula

-   -   in which, formula R₁, R₂, R₃, R₄ independently are a hydrogen        atom, a hydroxyl group or an alkoxy group with 1-5 carbon atoms;        each of R₅, R₆, R₇ is a hydroxyl group; and no more than 3 of        R₁, R₂, R₃, R₄ are hydrogen atoms;        precursors capable of liberating a substance according to the        aforementioned formula when used in the present method;        and mixtures of one or more of the aforementioned substances        and/or precursors. The term “oral administration” as used in        here also encompasses oral gavage administration.

The HRT method according to the invention may advantageously be used totreat all known forms of hypoestrogenism, e.g. hypoestrogenismassociated with (peri-)menopausal and post-menopausal women,hypoestrogenism resulting from hypogonadism or castration, as well ashypoestrogenism resulting from primary ovarian failure, treatment ofe.g. breast cancer with aromatase inhibitor and gonadotropin-releasinghormone analogue treatment of e.g. benign gynaecological diseases.Examples of manifestations of hypoestrogenism that can effectively betreated or prevented with the present method in both females and malesinclude osteoporosis, arteriosclerosis, cognitive disturbances andAlzheimer's disease. The method may also advantageously be used in the(prophylactic) treatment of climacteric symptoms such as hot flushes(flashes), sweats, urogenital atrophy, mood disturbances, insomnia andpalpitations. The present method is particularly suited for treating orpreventing osteoporosis and climacteric symptoms.

The term “estrogenic component” as used throughout this documentencompasses substances that are capable of triggering an estrogenicresponse in vivo, as well as precursors that are capable of liberatingsuch an estrogenic component in vivo when used in accordance with thepresent invention. In order for estrogenic components to trigger such aresponse they normally have to bind to an estrogen receptor, whichreceptors are found in various tissues within the mammalian body. Theterm “progestogenic component” is defined as a substance that is capableof triggering an progestogenic response in vivo or a precursor which iscapable of liberating such a substance in vivo. Usually progestogeniccomponents are capable of binding to a progestogen receptor.

It is noted that the present invention not only encompasses the use ofestrogenic and progestogenic components specifically mentioned in thisapplication, but also metabolites of these hormones that displaycomparable in vivo functionality. In this context it is observed that,for instance, levonorgestrel is a metabolite of norgestimate and thatestriol is a metabolite of 17beta-estradiol. Both these progestogens andestrogens have found application in contraceptive formulations and/orhormone replacement therapy. The term “estrogenic substances” as used inthis document does not encompass tritium (³H) labeled estrogenicsubstances such as tritium labeled estetrol.

The present estrogenic substances are distinct from both the biogenicand synthetic estrogens that are commonly applied in pharmaceuticalformulations in that they contain at least 4 hydroxyl groups. Thepresent substances are special in that the 5 membered ring in thesteroid skeleton comprises 3 hydroxyl substituents rather than 0-2.

Known estrogens that contain at least 4-hydroxyl groups and derivativesthereof are:

1,3,5(10)-estratrien-2,3,15α,16α,17β-pentol 2-methyl ether

1,3,5(10)-estratrien-2,3,15β,16α,17β-pentol 2-methyl ether

1,3,5(10)-estratrien-2,3,16α,17β-tetrol

1,3,5(10)-estratrien-3,4,16α,17β-tetrol 4-methyl ether

1,3,5(10)-estratrien-3,15α,16α,17β-tetrol

1,3,5(10)-estratrien-3,15α,16α,17β-tetrol tetra acetate

1,3,5(10)-estratrien-3,15β,16β,17β-tetrol tetra acetate

Preferably, the estrogenic substance applied as the active component inthe present composition is a natural estrogen, i.e. an estrogen that isfound in nature and especially in mammals. Even more preferably, theestrogenic substance is a so called biogenic estrogen, i.e. an estrogenthat occurs naturally in the human body, a precursor of a biogenicestrogen or mixtures thereof. Because biogenic estrogens are naturallypresent in the fetal and female body, side-effects are not expected tooccur, particularly not if the serum levels resulting from the exogenousadministration of such estrogens do not substantially exceed naturallyoccurring concentrations. Since estetrol serum levels in the fetus areseveral times higher than those found in pregnant females and knowingthat the fetus is particularly vulnerable, estetrol is deemed to be aparticularly safe biogenic estrogen. Side-effects are not expected tooccur, particularly not if the serum levels resulting from the exogenousadministration of such estrogens do not substantially exceed naturallyoccurring (fetal) concentrations. With synthetic estrogens such asethinyl estradiol there is a (dose dependent) risk of undesirableside-effects, such as thromboembolism, fluid retention, nausea,bloating, cholelithiasis, headache and breast pain.

In a preferred embodiment of the present invention the estrogenicsubstance contains 4 hydroxyl groups. Also, in the aforementionedformula, R₁ preferably represents a hydrogen atom. In said formulapreferably at least 2, more preferably at least 3 of the groups R₁, R₂,R₃ and R₄ represent a hydrogen atom.

The estrogenic substances according to the formula encompass variousenantiomers since the carbon atoms that carry hydroxyl-substituents R₅,R₆ and R₇ are chirally active. In one preferred embodiment, the presentestrogenic substance is 15α-hydroxy substituted. In another preferredembodiment the substance is 16α-hydroxy substituted. In yet anotherpreferred embodiment, the substances is 17β-hydroxy substituted. Mostpreferably the estrogenic substances are 15α,16α,17β-trihydroxysubstituted.

In another preferred embodiment of the present invention R₃ represents ahydroxyl group or an alkoxy group. In another preferred embodiment thegroups R₁, R₂ and R₄ represent hydrogen atoms, in which case, if R₃, R₅,R₆ and R₇ are hydroxyl groups, the substance is1,3,5(10)-estratrien-3,15,16,17-tetrol. A preferred isomer of the lattersubstance is 1,3,5(10)-estratrien-3,15α,16α,17β-tetrol (estetrol).

The invention also encompasses the use of precursors of the estrogenicsubstances that constitute the active component in the present method.These precursors are capable of liberating the aforementioned estrogenicsubstances when used in the present method, e.g. as a result ofmetabolic conversion. These precursors are preferably selected from thegroup of androgenic precursors as well as derivatives of the presentestrogenic substances. Suitable examples of androgenic precursorsinclude androgens that can be converted into the present estrogenicsubstances through in vivo aromatisation. Examples of derivatives of thepresent estrogenic substances that can suitably be used as precursorsinclude such substances wherein the hydrogen atom of at least one of thehydroxyl groups has been substituted by an acyl radical of a hydrocarboncarboxylic, sulfonic acid or sulfamic acid of 1-25 carbon atoms;tetrahydrofuranyl; tetrahydropyranal; or a straight or branched chainglycosidic residue containing 1-20-glycosidic units per residue.

Typical examples of precursors which can suitably be used in accordancewith the invention are esters that can be obtained by reacting thehydroxyl groups of the estrogenic substances with substances thatcontain one or more carboxy (M⁺⁻OOC—) groups, wherein M⁺ represents ahydrogen or (akali)metal cation. Hence, in a particularly preferredembodiment, the precursors are derivatives of the estrogenic substances,wherein the hydrogen atom of at least one of the hydroxyl groups in saidformula has been substituted by —CO—R, wherein R is a hydrocarbonradical comprising from 1-25 carbon atoms. Preferably R is hydrogen, oran alkyl, alkenyl or aryl radical comprising from 1-20 carbon atoms.

The present method usually employs uninterrupted oral administration ofthe estrogenic component during a period of at least 10 days, preferablyof at least 20 days. The term “uninterrupted” as used in here, meansthat the estrogenic component is administered at relatively regularintervals, with no (therapeutically) significant interruptions.Naturally, minor interruptions may occur that do not affect the overalleffectiveness of the present method, and indeed such aberrations areencompassed by the present invention. In a preferred embodiment, andmore arithmetically, the administration regimen is deemed to becontinuous if the longest interval between 2 subsequent administrationsis not more than 3.5 times as long as the average interval. Even morepreferably said longest interval is not more than 2.5 times, mostpreferably not more than 1.5 times as long as the average interval.

The benefits of the present invention are most pronounced when theestrogen component is used in longer term hormone replacement therapy soas to minimise the negative effects of chronic hypoestrogenism.Therefore, the method of hormone replacement therapy, preferably,comprises administering the estrogenic component for a period of atleast 1 month, more preferably of at least 3 months.

In the present method, the estrogenic and progestogenic component may beadministered in separate oral dosage units. However, it is also possibleand indeed very convenient to combine these two components into a singleoral dosage unit.

The invention may suitably be reduced to practice in the form of avariety of HRT methods that are known to the person skilled in the art.Amongst these methods are the so called “combined” methods. The combinedmethods make use of preparations that contain a combination of anestrogen and a progestogen. The combined methods have in common thatthey are based on a regimen which involves administration of theaforementioned combined preparation, followed by an administration-freeinterval of about 7 days whereby withdrawal bleeding, simulating thenatural menses, occurs. Thus 21 day intervals of hormone administrationalternate with 7 days during which no hormones are administered.

As an alternative to the aforementioned combined methods, the so called“sequential” method has been proposed. Typical of the sequential methodis that it comprises two consecutive phases, i.e. one phase during whichestrogen and no progestogen is administered and another phase duringwhich a combination of estrogen and progestogen is administered. Thefirst sequential methods, like the aforementioned combined methods, madeuse of an administration free interval of about 7 days. More recently,sequential methods have been proposed which do not include anadministration-free (or placebo) period, meaning that estrogen isadministered throughout the full cycle and that progestogen isco-administered during only part of that cycle. WO 95/17895 (Ehrlich etal.) describes such an uninterrupted sequential method.

Yet another example of an HRT method which is encompassed by the presentinvention is the so called “continuous combined” method, which is aparticular version of the combined method that uses uninterruptedcombined administration of a progestogenic and an estrogenic componentduring a prolonged period of time, e.g. more than 50 days. In contrastto ordinary combined and sequential methods, no regular menses occur inthe continuous combined method as the continuous administration ofprogestogen in the indicated amounts induces amenorrhoea.

In one embodiment of the invention, which relates to the continuouscombined method, the present method comprises the uninterrupted oraladministration of the combination of the estrogenic component and theprogestogenic component during a period of at least 28, preferably atleast 60 days.

In another embodiment of the invention, which relates to sequential andcombined methods that employ a significant administration-free interval,the method of the invention comprises an interval of at least 2 days,preferably from 3-9 days, most preferably from 5-8 days, during which noprogestogenic component and no estrogenic component is administered andwherein the resulting decrease in serum concentration of theprogestogenic component and the estrogenic component induces menses.

Yet another embodiment of the invention, which concerns a sequentialmethod without a significant pause, is characterised in that itcomprises the uninterrupted oral administration of the estrogeniccomponent during a period of at least 28 days, preferably at least 60days, and in that, following the combined administration of theestrogenic component and the progestogenic component, the estrogeniccomponent and no progestogenic component are administered during 3-18consecutive days, preferably during 5-16 consecutive days and theresulting decrease in serum concentration of the progestogenic componentshould normally be sufficient to induce menses.

In the present methods uninterrupted administration of the estrogeniccomponent may usually occur at intervals of between 6 hours and 7 days,preferably of between 12 hours and 3 days. The relatively high in vivohalf-life of the present estrogenic components in comparison to mostknown estrogens makes it feasible to employ oral administrationintervals that are significantly longer than 1 day. For practicalreasons, and particularly with a view to user compliance, it ispreferred to orally administer the estrogenic component as well as theprogestogenic component at least once daily, most preferably once daily.

In all of the aforementioned methods it is preferred to orallyadminister the estrogenic component and the progestogenic component atleast once daily during a period of at least 10, preferably of at least20 days. In case of a sequential method without pause or a continuouscombined method it is preferred to orally administer the estrogeniccomponent and/or the progestogenic component at least once daily duringa period of at least 30 days, more preferably of at least 60 days, mostpreferably of at least 150 days. Uninterrupted sequential methods, whichemploy continuous estrogen administration, are characterised byexcellent cycle control.

The general concerns about the so called unopposed administration ofestrogen, i.e. administration of estrogen without co-administeredprogestogen might cause hyperplasia of the endometrium, are lessapplicable to the estrogenic components of the present invention.Therefore, in a particularly preferred embodiment, the present HRTmethod is executed in accordance with a sequential method without pause.

Good results can be obtained with the present method if the estrogeniccomponent is orally administered in an amount of less than 1 mg per kgof bodyweight per day, preferably of less than 400 μg per kg ofbodyweight per day, more preferably of less than 200 μg per kg ofbodyweight per day. In order to achieve a significant impact from theadministration of the present estrogenic component, it is advisable toorally administer in an amount of at least 1 μg per kg of bodyweight perday. Preferably, the orally administered amount is at least 2 μg per kgof bodyweight per day. More preferably, the orally administered amountis at least 5 μg per kg of bodyweight per day.

In the present method, particularly when used in humans, the estrogeniccomponent is usually administered in an average dosage of at least 0.05mg per day, preferably of at least 0.1 mg per day. The maximum dosage isnormally kept below 40 mg per day, preferably below 20 mg per day. Thenormally employed dose of the progestogenic component is equivalent toan average oral dosage of 30-750 μg levonorgestrel per day, preferablyto an average oral dosage of 50-400 μg levonorgestrel per day.

In the present method, the estrogenic component is preferablyadministered in an amount effective to achieve a blood serumconcentration of at least 1 nanogram per liter, more preferably of atleast 10 nanogram per liter, most preferably at least 100 nanogram perliter. Generally the resulting blood serum concentration of theestrogenic component will not exceed 100 μg per liter, preferably itwill not exceed 50 μg per liter, more preferably it will not exceed 25μg per liter.

In accordance with the present invention the progestogenic component isadvantageously administered in an amount which is equivalent to a dailyoral dosage of 0.3 to 20 μg levonorgestrel per kg of bodyweight,preferably of 0.5-5 μg levonorgestrel per kg of bodyweight.

Examples of progestogens which may suitably be used in accordance withthe present invention include: progesterone, levonorgestrel,norgestimate, norethisterone, dydrogesterone, drospirenone,3-beta-hydroxydesogestrel, 3-keto desogestrel (=etonogestrel),17-deacetyl norgestimate, 19-norprogesterone, acetoxypregnenolone,allylestrenol, anagestone, chlormadinone, cyproterone, demegestone,desogestrel, dienogest, dihydrogesterone, dimethisterone, ethisterone,ethynodiol diacetate, fluorogestone acetate, gastrinon, gestodene,gestrinone, hydroxymethylprogesterone, hydroxyprogesterone, lynestrenol(=lynoestrenol), medrogestone, medroxyprogesterone, megestrol,melengestrol, nomegestrol, norethindrone (norethisterone),norethynodrel, norgestrel (includes d-norgestrel and dl-norgestrel),norgestrienone, normethisterone, progesterone, quingestanol,(17alpha)-17-hydroxy-11-methylene-19-norpregna-4,15-diene-20-yn-3-one,tibolone, trimegestone, algestone acetophenide, nestorone, promegestone,17-hydroxyprogesterone esters, 19-nor-17hydroxyprogesterone,17alpha-ethinyl-testosterone, 17alpha-ethinyl-19-nor-testosterone,d-17beta-acetoxy-13beta-ethyl-17alpha-ethinyl-gon-4-en-3-one oxime andprecursors of these compounds that are capable of liberating theseprogestogens in vivo when used in the present method. Preferably theprogestogen used in the present method is selected from the groupconsisting of progesterone, desogestrel, etonogestrel, gestodene,dienogest, levonorgestrel, norgestimate, norethisterone, drospirenone,trimegestone, dydrogesterone, precursors of these progestogens andmixtures thereof.

The present method also encompasses the co-administration of activeprinciples in addition to the progestogenic and estrogenic component.For instance, androgens may advantageously be co-administered in orderto prevent symptoms of hypoandrogenicity. Thus, a preferred embodimentof the invention comprises the co-administration of an androgeniccomponent. The androgenic component is suitably co-administered in aneffective amount to suppress symptoms of hypoandrogenicity.Hypoandrogenicity has been associated with mood disturbances,unfavourable changes in haemostatic parameters and lack of bone mass.

The term “androgenic component” is defined as a substance that iscapable of triggering an androgenic response in vivo or a precursorwhich is capable of liberating such a substance in vivo. Usuallyandrogenic components are capable of binding to an androgen receptor.

Androgenic components that may suitably be employed in the presentmethod may be selected from the group consisting ofdehydroepiandrosterone (DHEA), danazol, gestrinone, testosterone esters,precursors capable of liberating these androgens when used in thepresent method and mixtures thereof. Preferably the testosterone estersemployed comprise an acyl group which comprises at least 6, morepreferably from 8-20 and preferably 9-13 carbon atoms. The androgensthat can be used most advantageously in the present method are DHEAand/or testosterone undecanoate.

It is noted that, for instance, DHEA and testosterone undecanoate areprecursors of testosterone and that said precursors per se exhibitvirtually no affinity for androgen receptors in the female body. Theeffectiveness of the androgens within the method of the invention isdetermined by their functionally active form, which may well bedifferent from the form in which they are administered.

In a preferred embodiment the androgen is provided in an amountequivalent to a daily oral dosage of 5 to 250 mg DHEA, which isequivalent to a daily oral dosage of 1 to 50 mg testosteroneundecanoate. More preferably the androgen is provided in an amount whichis equivalent to a daily oral dosage of 10-120 mg DHEA. Most preferablythe androgen is administered in an amount which is equivalent to a dailyoral dosage of 20-60 mg DHEA.

In order to obtain the desired impact from the present method it isadvisable to administer the dosage units in an amount which leads to anincrease in blood serum androgen level of no more than 5 nmoletestosterone equivalent per liter, preferably less than 3 nmoletestosterone equivalent per liter and most preferably less than 1.5nmole testosterone equivalent per liter.

The present method preferably does not employ a gonadotropin hormonereleasing hormone composition as described in the aforementioned patentsU.S. Pat. No. 5,211,952, U.S. Pat. No. 5,340,584 and U.S. Pat. No.5,340,585. Similarly, the present method preferably does not employ aluteinizing hormone releasing hormone composition as described in U.S.Pat. No. 4,762,717 and U.S. Pat. No. 5,130,137. Furthermore, the presentmethod preferably does not comprise the co-administration of ananti-progestogen as described in U.S. Pat. No. 5,468,736. The method mayalso suitably be applied without the co-administration of an antisenseoligonucleotide that is complementary to the nucleotide sequence of thefollicle stimulating hormone (FSH) receptor (WO 00/73416).

The present method is preferably not used in oophorectomised females orin females in whom endometrial stimulation by estrogenic compositions isminimised or absent, other than by combined administration of aprogestogen and an estrogen, e.g. as a result of hysterectomy.

Another aspect of the invention relates to a pharmaceutical kitcomprising at least 20 oral dosage units that contains the estrogeniccomponent as defined herein before and/or the progestogenic componentand/or the androgenic component as described herein before, wherein atleast 10 units contain between 0.01 and 20 mg, preferably between 0.05and 10 mg of the estrogenic component, at least 10 units contain theprogestogenic component in an amount equivalent to 30-750 μglevonorgestrel and at least 10 dosage units contain the androgeniccomponent in an amount equivalent to 5-250 mg dehydroepiandrosterone.

In the present kit, the estrogenic component may conveniently becombined with the progestogenic component and the androgenic in a singledosage unit. Accordingly, the kit preferably comprises at least 10dosage units which contain between 0.01 and 20 mg of the estrogeniccomponent, the progestogenic component in an amount equivalent to 30-750μg levonorgestrel and the androgenic component in an amount equivalentto 5-250 mg dehydroepiandrosterone. A pharmaceutical kit that isparticularly suitable for use in combined and sequential methods willusually comprise 20-35 oral dosage units, wherein 10-35 units contain acombination of the estrogenic component and the progestogenic componentin the indicated amounts, 0-25 units contain no progestogenic componentand the estrogenic component in the indicated amounts, and 0-8 unitscontain no estrogenic component and no progestogenic component.

A pharmaceutical kit that is particularly suitable for use in acontinuous combined regimen or a combined regimen comprises at least 20oral dosage units which either contain the combination of theprogestogenic and the estrogenic component or neither of these twocomponents (placebo's) and of which dosage units at least 15, preferablyat least 20 contain the combination of the estrogenic component and theprogestogenic component and 0-8 contain no estrogenic component and noprogestogenic component. If such a kit is to be used in a continuouscombined method, the kit may advantageously comprise at least 28,preferably at least 60 dosage units, all of which dosage units containthe combination of the estrogenic component and the progestogeniccomponent in the amounts indicated above.

In case the present kit is meant to be used in an HRT method thatemploys an administration free interval so as to induce menses (e.g. acombined method or a sequential method with pause) the kit will usuallycomprise at least 3 units, preferably at least 5 units that contain noestrogenic component and no progestogenic component.

In a particularly preferred embodiment of the invention the present kitis designed for use in a sequential method. Such a kit will usuallycomprise 20-35 oral dosage units wherein 10-32 units contain thecombination of the estrogenic component and the progestogenic component,and 3-18 units contain the estrogenic component and no progestogeniccomponent. Particularly preferred is a kit that is designed for use in asequential method without a significant pause. In such a kit, which willusually comprise 20-35 oral dosage units, 10-20 units contain acombination of the estrogenic component and the progestogenic component,10-18 units contain the estrogenic component and no progestogeniccomponent and at most 1 unit contains no estrogenic component and noprogestogenic component.

The pharmaceutical kits according to the present invention will normallycontain only one or more of the following types of oral dosage units:units that contain the combination of the estrogenic and theprogestogenic component; units that contain the estrogenic component andno progestogenic component; and units that effectively function asplacebo's. Preferably the kit comprises at least 20 units that containthe combination of the estrogenic and the progestogenic component or theestrogenic component and no progestogenic component.

If the present kit is to be used in a combined or sequential protocol,the oral dosage units are preferably arranged within the kit in a fixedsequence corresponding to the intended order of administration. Dataindications may be provided on the packaging. The packaging may be atube or box or a strip. The box may be circular, square, or otherwiseshaped with the tablets being accommodated separately therein for easeof administration. Date indications may appear adjacent to each tabletcorresponding with the days on which each tablet is to be taken. Someindication of the sequence in which the tablets are to be takenpreferably appears on the packaging regardless of its form.

Generally speaking, the oral dosage units in the present kit areprepared according to well known pharmaceutical procedures. The activeingredient(s) are combined with a pharmaceutically acceptable excipientand converted into a pharmaceutically acceptable form for oraladministration, e.g. a tablet, capsule, cachet, pellet, pill, powder orgranules. The excipient may include appropriate pharmaceutical carrierssuch as diluents, binders and lubricants. For example gums, starches andsugars are commonly used as pharmaceutical carriers. Tablets and otheroral dosage units can suitably contain materials such binders (e.g.hydroxypropylmethyl cellulose, polyvinyl pyrrolidine, other cellulosicmaterials and starch), diluents (e.g. lactose and other sugars, starch,dicalcium phosphate and cellulosic materials), disintegrating agents(e.g. starch polymers and cellulosic materials) and lubricating agents(e.g., stearates and talc).

The active ingredient(s) may comprise from about 0.01% by weight toabout 50% by weight of the formulation in the dosage unit, the remainderconsisting of excipient. The active ingredient(s) are compounded withthe chosen carrier and in for example the case of a tablet form, placedin a tablet moulding apparatus to form the tablets. Alternatively thecompounded material may be incorporated as a powder or granules in acapsule. Various other options that may suitably be used in accordancewith the present invention are well known to the person skilled in thepharmaceutical art.

The present invention is further illustrated by the following examples,which, however, are not to be construed as limiting. The featuresdisclosed in the foregoing description, in the following examples and inthe claims may, both separately and in any combination thereof, bematerial for realising the invention in diverse forms thereof.

EXAMPLES Example 1

Vaginal cornification was chosen as a tissue-specific andestrogen-sensitive endpoint to determine the estrogenicity of estetrol(E4), after oral administration, in hypoestrogenic rats.17α-ethinylestradiol (EE) and vehicle (10% ethanol/sesame oil) served ascontrols in these bioassays.

Uterine weight increase in the rat is more commonly used as a measure ofestrogenicity. However, uterine weight also responds to progesterone,testosterone, and other agents not characteristically regarded asestrogens. In the early 1920s it was discovered that follicular fluidfrom the pig ovary contained a factor(s) that causedcornification/keratinization of the vaginal epithelium in the rat (Allenand Doisy, 1923, JAMA, 81, 819-821; Allen and Doisy, 1924, Am. J.Physiol., 69, 577-588). The so-called vaginal cornification response inrats subsequently provided a bioassay for testing estrogenicity. Vaginalepithelial cornification/keratinization in ovariectomized rats can beproduced only by compounds considered to be true estrogens (Jones et al,1973, Fert. Steril. 24, 284-291). Vaginal epithelialcornification/keratinization represents, therefore, a highly selectiveendpoint to determine the potency of estrogens (Reel et al., 1996, Fund.Appli. Toxicol. 34, 288-305).

Adult intact female CD rats were ovariectomized to induce estrogendeficiency. Vaginal lavages were performed daily for seven days toensure that the rats demonstrated castrate vaginal smears (predominanceof leukocytes in the vaginal smear, and similar in appearance to adiestrous vaginal smear). Castrate vaginal smears are indicative thatcomplete ovariectomy was achieved. Treatment commenced followingcompletion of the 7 days of smearing (day 0=first day of dosing).Animals were dosed, once daily for 7 consecutive days. Daily vaginallavages continued to be obtained for 7 days after dosing was initiatedin order to detect vaginal cornification, as an indication of anestrogenic response. A drop of vaginal washings was placed on a glassslide and examined by light microscopy to detect the presence or absenceof cornified epithelial cells. Vaginal lavages were obtained prior todosing on days 0-6 and prior to necropsy on day 7.

The vaginal cornification bioassay was performed in order to determinethe estrogenic profile of E4 when given orally (po) to ovariectomizedadult rats. EE was used as a positive control. The vehicle (10%ethanol/sesame oil) served as the negative control. Steroids weredissolved in absolute ethanol and then brought to the finalconcentration with sesame oil (10% ethanol in sesame oil). A vaginalestrogenic response occurred in all rats (8/8) given 50 μg/kg/day EE poby day 7 (Table 1). Similarly, vaginal epithelial cornification wasobserved in all rats (8/8) treated po with either 0.1, 0.3, 1.0, or 3.0mg/kg/day E4 by day 7 (Table 1), whereas animals treated with thevehicle did not exhibit vaginal epithelial cornification (0/8). Even inrats given relatively low doses of E4 (e.g. 0.1 and 0.3 mg/kg/day), theonset of vaginal cornification (defined as the amount of animalsresponding at days 1-3 of the study) was as fast as in EE-treatedanimals (Table 1), demonstrating estetrol's superb bioavailabilitycharacteristics after oral administration.

Table 1: Vaginal estrogenic response in ovariectomized rats treatedorally (po) with 17α-ethinyl estradiol (EE) or estetrol (E4). Data areexpressed as the number of rats showing vaginal cornification over thenumber of rats (ratio) treated.

Number of Rats Exhibiting Estrogenic Response/Number of Rats TreatedDosing Day of Study Treatment Group route Day 0 Day 1 Day 2 Day 3 Day 4Day 5 Day 6 Day 7 0.05 mg/kg/day EE po 0/8 1/8 3/8 8/8 8/8 8/8 8/8 8/8Vehicle po 0/8 0/8 0/8 0/8 0/8 0/8 0/8 0/8 Control (2 ml/kg/day) 0.1mg/kg/day E4 po 0/8 0/8 1/8 7/8 8/8 8/8 8/8 8/8 0.3 mg/kg/day E4 po 0/80/8 1/8 7/8 8/8 8/8 8/8 8/8 1.0 mg/kg/day E4 po 0/8 0/8 4/8 8/8 8/8 8/88/8 8/8 3.0 mg/kg/day E4 po 0/8 0/8 6/8 8/8 8/8 8/8 8/8 8/8

Example 2

The ovariectomized aged rat was used as a model for the human diseaseosteoporosis. This is an established animal model, recommended by theUnited States Food and Drug Administration (FDA), to evaluate and assesspotential agents for osteoporosis prevention and therapy. Theanti-resorptive efficacy of estetrol (E4) was tested by ex vivomeasuring total and trabecular bone mineral density and bone strengthafter 4 weeks of treatment at necropsy. 17α-ethinyl-estradiol (EE) andvehicle (1% ethanol/arachidis oil) served as controls in this bioassay.

Three months old female Sprague-Dawley rats were either sham-operated(Sham) or ovariectomized (OVX) one day prior to commencement of thedosing study. Animals were anesthetized using a ketamine/xylazineanesthetic mixture and underwent a bilateral ovariectomy or weresham-treated. A section of hair on the dorsal surface was shaved and anincision made overlying the lumbar region of the spine. The skin wasseparated from the underlying fascia so that a second incision could bemade through the abdominal musculature approximately caudal to thekidneys. The ovaries were then exteriorized and removed and themusculature was closed with a single suture. The skin incision wasclosed using surgical staples.

Ten animals per treatment group were orally dosed once per day for fourconsecutive weeks. The dosing commenced 1 day after surgical removal ofthe ovaries and was administered by oral gavage using a syringe andstainless steel gavage needle at doses of 0.1 mg/kg/day EE, or either2.5, 0.5 or 0.1 mg/kg/day E4. Vehicle control was daily administered toone group of OVX-animals and sham-operated rats. After treatment,anesthetized rats were subjected to cardiac puncture and asphyxiated byCO₂ inhalation. Tibiae and femura were removed, cleaned of soft-tissueand fixed and stored in 70% ethanol/saline at 4° C. (tibia) or saline at4° C. (femura) until further analysis.

Ex vivo peripheral Quantitative Computed Tomography (pQCT) was performedon the excised left tibiae using a Stratec XCT-RM and associatedsoftware (Stratec Medizintechnik GmbH, Pforzheim, Germany, softwareversion 5.40). The scans were performed at 12% of the total length fromthe proximal end of the tibiae. The positions were verified using scoutviews and one 0.5-mm slice perpendicular to the long axis of the tibialshaft was acquired from each site. The scans were analyzed using athreshold for delineation of the external boundary. The total andtrabecular bone mineral content, area and density at each site weredetermined. Mean values are shown in Table 2. Furthermore, pQCT data formean total bone mineral density are depicted in FIG. 1.

Table 2: pQCT densitometry data from the proximal tibiae of Sham- andOVX-rats orally (po) treated with 17α-ethinyl estradiol (EE), estetrol(E4) or vehicle. Data are expressed as the mean values obtained for eachgroup (n=10).

Mean Total Mean Trabecular Treatment Bone Mineral Bone Mineral GroupDosing Content Area Density Content Area Density (n = 10) route (mg/mm)(mm²) (mg/cm³) (mg/mm) (mm²) (mg/cm³) SHAM + po 9.36 14.10 664.07 1.496.34 235.48 Vehicle OVX + po 8.76 14.47 606.61 1.10 6.51 169.63 VehicleOVX + 0.1 po 9.66 13.87 697.48 1.81 6.24 290.16 mg/kg/day EE OVX + 0.1po 8.46 14.41 588.62 0.96 6.48 145.46 mg/kg/day E4 OVX + 0.5 po 9.7414.80 660.57 1.60 6.65 243.31 mg/kg/day E4 OVX + 2.5 po 9.61 13.59707.11 1.89 6.12 309.58 mg/kg/day E4

Comparison of the pQCT densitometry data from the proximal tibiae ofSham-operated and OVX-rats demonstrated a consistent loss of total andtrabecular bone in the OVX-group, as expected (Table 2, FIG. 1).Furthermore, there was a consistent dose-dependent increase for each ofthe parameters associated with total and trabecular bone mineral contentand bone mineral density in the animals orally treated with E4 (Table 2,FIG. 1). As compared to hypoestrogenic OVX-rats receiving vehicletreatment alone, 0.5 and 2.5 mg/kg/day E4 prevented bone resorption asexemplified by total bone mineral density levels equivalent tosham-operated rats (FIG. 1). Furthermore, the anti-resorptive activityachieved with the highest dose of E4 (2.5 mg/kg/day) was equivalent tothe effect seen with the positive control EE.

Ex vivo evaluation of bone biomechanical strength was performed with anindentation test at the distal femura. Prior to mechanical testingfemura were rinsed in cold saline and carefully cleaned of any remainingadherent soft tissue. A 3-mm segment of the distal femoral metaphysiswas cut directly proximal to the femoral condyle with a low-speeddiamond saw under constant saline irrigation. The load was applied witha cylindrical indenter (with a flat testing face of 1.6 mm diameter) tothe center of marrow cavity on the distal face of the segment. Theindenter was allowed to penetrate the cavity at a constant displacementof 6 mm/min to a depth of 2 mm before load reversal.

Table 3: Indentation testing of the distal femur of Sham- and OVX-ratsorally (po) treated with 17α-ethinyl estradiol (EE), estetrol (E4) orvehicle. Data are expressed as the mean values obtained for each group(n=10).

Treatment Maximum Ultimate Group Dosing load Stiffness Energy strength(n = 10) route (N) (N/mm) (mJ) (N/mm2) SHAM + po 8.61 131.96 0.48 4.57Vehicle OVX + po 2.77 42.08 0.21 1.47 Vehicle OVX + 0.1 po 9.05 169.120.53 4.80 mg/kg/day EE OVX + 0.1 po 1.50 28.00 0.09 0.80 mg/kg/day E4OVX + 0.5 po 7.25 132.57 0.31 3.85 mg/kg/day E4 OVX + 2.5 po 13.07173.12 0.68 6.94 mg/kg/day E4

The maximum load, stiffness and energy absorbed were determined fromload displacement curves. Ultimate strength was calculated by dividingthe maximum load by the indenter area. Mean values of maximum load,stiffness, energy and ultimate strength are shown in Table 3.Furthermore, mean ultimate strength values are depicted in FIG. 2. Ascompared to Sham-operated rats, the mechanical strength of cancellousbone appeared to be markedly reduced in OVX-rats treated with vehiclealone (Table 3, FIG. 2). Reductions in maximum load, stiffness, energyand ultimate strength were −68%, −68%, −27% and −68%, respectively,clearly accompanying the bone mineral density loss in estrogen deficientrats. Oral treatment of hypoestrogenic OVX-rats with E4 prevented thedeclines in maximum load, stiffness, energy and ultimate strength, in adose-dependent manner (Table 3, FIG. 2). In addition, the efficacyachieved with the highest doses of E4 (2.5 mg/kg/day) even appearssuperior to that of the positive control EE (Table 3, FIG. 2).

Example 3

The morphine-dependent ovariectomized (OVX) rat was used as a model forpostmenopausal hot flush. The potency of estetrol (E4) to prevent tailskin temperature rises, normally accompanied by a drop in core bodytemperature, after naloxone-induced opiate withdrawal was tested.17α-ethinyl-estradiol (EE) and vehicle (hydroxy propyl-beta-cyclodextrin20% wt/vol) served as controls in this bioassay.

The most common and characteristic symptom of human menopause is the hotflush, which is experienced by over 70% of menopausal females. While theexact mechanism underlying this vasomotor instability is unknown, thecharacteristic features of the hot flush appear to reflect a centrallymediated adaptation to a progressive decline in the levels of estrogens.In women experiencing the hot flush the symptoms are manifested by 1)rapid, regional elevations in skin temperature; 2) a decrease in corebody temperature; 3) an increased heart rate with no change in bloodpressure; and 4) closely timed surges in release of luteinizing hormone(LH) and β-endorphin.

The morphine-dependent ovariectomized (OVX) rat model has been proposedby several investigators (Katovich et al, 1986, Maturitas, 67-76;Merchenthaler et al. 1998, Maturitas, 307-316) as an animal model forthe hot flush. During opiate withdrawal with the morphine antagonistnaloxone, tail skin temperature (TST) rises and this rise is accompaniedby a drop in core body temperature. In addition, the temperature changesare accompanied by surges in LH and a transient tachycardia. Theseevents are similar in magnitude and temporal nature to those observed inthe menopausal hot flush.

8-week-old OVX rats were treated orally (po) with estetrol (E4),17α-ethinyl estradiol (EE) or vehicle control (hydroxypropyl-beta-cyclodextrin 20% wt/vol) for seven consecutive days priorto, and on the morning of naloxone-induced opiate withdrawal inmorphine-dependent animals. Three days prior to the commencement ofdosing, animals were anesthetized using a ketamine/xylazine anestheticmixture and underwent a bilateral ovariectomy. A section of hair on thedorsal surface was shaved and an incision made overlying the lumbarregion of the spine. The skin was separated from the underlying fasciaso that a second incision could be made through the abdominalmusculature approximately caudal to the kidneys. The ovaries were thenexteriorized and removed and the musculature was closed with a singlesuture. The skin incision was closed using surgical staples. Six ratsper treatment group were dosed once per day for eight consecutive daysprior to and including the day of naloxone-induced opiate withdrawal(the hot flush session). The dosing commenced three days after surgicalremoval of the ovaries and was administered by oral gavage using asyringe and stainless steel gavage needle. Morphine dependency wasinduced by implantation of subcutaneous pellets containing 75-mgmorphine. The first pellet was implanted five days before the hot flushsession under a light inhalation anesthesia. Three days before the hotflush session, two additional morphine pellets were implanted under thesame conditions.

For the hot flush manipulations the animals were placed in a test cage.Following a 5-10 minute adaptation period, the rats were anesthetizedwith ketamine HCl approximately 10 minutes prior to the hot flushsession. A temperature sensitive electrode was fixed to the ventralsurface of the tail with tape and the electrode was connected to amulti-channel temperature recorder. The tail-skin temperature wasrecorded until it was stable and the animals were then injected withnaloxone HCl (1 mg/kg). The temperature recordings then continued for aperiod of 60 minutes and the temperature was reported at 5-minuteintervals. At the completion of the hot flush session, all animals werekilled using CO₂ asphyxiation followed by cervical dislocation.

As expected, vehicle control was ineffective in preventing thenaloxone-induced TST increases in the morphine addicted OVX rats (FIG.3). 17α-ethinyl estradiol (EE), at the single dose tested of 0.3mg/kg/day, prevented the naloxone-induced TST increases in the morphineaddicted OVX rats (FIG. 3). Oral treatment with estetrol (E4) showed aclear dose-dependent effect (FIG. 3). The three highest doses of E4(0.3, 1.0 and 3.0 mg/kg/day) all attenuated the TST, with the highestdose (3.0 mg/kg/day) having a suppressive response similar to the potentoral estrogen, 17α-ethinyl estradiol (EE).

Example 4

To evaluate the oral bioavailability of estetrol (E4) and to determinethe elimination half-life, single oral (po) and subcutaneous (sc) dosestudies were performed in female Sprague Dawley rats followed byfrequent blood sampling over a 24 hours interval.

Female Sprague Dawley rats were equipped with a permanent silatic heartcatheter, as described by Kuipers et al. (1985, Gastroenterology, 88,403-411). Rats were allowed to recover from surgery for 5 days and werethan administered 0.05, 0.5, or 5 mg/kg E4 in 0.5 ml arachidis oil. Forsc administration, E4 was injected in the neck area using a 1 ml syringeand 20 g needle. For po administration of E4, rats were lightlyanaesthesized with halothene/N₂O/O₂ and E4 was directly appliedintragastrically using a plastic stomach intubator. Blood samples weresubsequently collected via the heart catheter in heparinized tubes at0.5, 1, 2, 4, 8 and 24 hours. Erythrocytes were removed bycentrifugation at 5000×g for 10 minutes at 4° C. and blood plasma wasstored at −20° C. After thawing the plasma samples, liquid-liquidextraction (hexane and diethyl ether) was employed to prepare theE4-containing plasma samples for HPLC analysis (Perkin Elmer 200) andtandem mass spectrometry using a PE Sciex 3000 tandem mass spectrometerand APCI interface. With each sample batch, a calibration curve with 6calibrators was recorded. The calibration curve was calculated usinglinear regression (correlation coefficient >0.98), which permittedquantitation of plasma concentrations. For each rat plasma, sampled atdifferent time intervals, data were collected.

Plasma E4 concentration data were analysed with “WinNonLin, edition 3.1”and involved pharmacokinetic parameters for C_(max), half-life andAUC₀₋₂₄. Especially, using the lower and intermediate dose levels of0.05, 0.5 mg/kg, E4 demonstrated an oral bioavailability equal to thebioavailability obtained with sc administration (80-100%). At thehighest dose level tested, 5.0 mg/kg E4, absorption kinetics gave riseto an oral bioavailability approximating 30-60% of sc administered E4.Interestingly, E4 demonstrated a relatively long half-life of 2-3 hours,enabling the detection of bioactive levels of unconjugated E4 at alltime points over a 24 hour interval.

Example 5

An established competitive steroid-binding assay (Hammond andLahteenmaki. 1983. Clin Chem Acta 132:101-110) was used to determine therelative binding affinity of estetrol (E4), 17α-ethinylestradiol (EE2),17β-estradiol (E2), testosterone (T) and 5α-dihydrotestosterone (DHT)for human sex Hormone Binding Globulin (SHBG).

Human SHBG was purified from transgenic mouse serum, as describedpreviously (Avvakumov G V et al., 2000. J Biol Chem 275: 25920-25925).The human SHBG prepared in this way was assessed to be >99% pure bypolyacrylamide gel electrophoresis under denaturing conditions. Itssteroid-binding characteristics are indistinguishable from SHBG in humanserum (Avvakumov G V et al., 2000. J Biol Chem 275: 25920-25925). The invitro assay involved the use of the purified human SHBG and [³H]DHT or[³H]estradiol as labeled ligands. Human SHBG was treated for 30 min atroom temperature with a dextran-coated charcoal (DCC) suspension inphosphate buffered saline (PBS) to remove any steroid ligand. Aftercentrifugation (2,000×g for 10 min) to sediment the DCC, the supernatantcontaining the human SHBG was diluted in PBS to a concentration of 1 nMbased on its steroid binding capacity.

Duplicate aliquots (100 μl) of this human SHBG solution were thenincubated with an equal volume of either [³H]DHT or [³H]estradiol at 10nM, together with 100 μl of PBS alone or the same amount of PBScontaining increasing concentrations of unlabeled steroid ligands ascompetitors in polystyrene test tubes. After incubation for 1 h at roomtemperature the reaction mixtures were placed in an ice bath for afurther 15 min. Aliquots (600 μl) of an ice cold suspension of DCC werethen added to each tube, and after a brief 2 seconds mixing, each tubewas incubated in an ice bath for either 10 min or 5 min depending onwhether [³H]DHT or [³H]estradiol were being used as labeled ligands,respectively. The unbound ligands adsorbed to DCC were then removed bycentrifugation (2,000×g for 15 min at 4 C), and the amounts of[³H]labeled ligands bound to SHBG were counted in 2 ml ACS scintillationcocktail using in liquid scintillation spectrophotometer. The averageamounts of [³H]labeled ligands bound to SHBG at each concentration ofcompetitor (B) were expressed as a percentage of the average amounts of[³H]labeled ligands bound to SHBG in the absence of competitor (B₀), andwere plotted against the concentration of competitor in each assay tube.

The results of the competitive binding assays are depicted in FIG. 4. Asis clearly apparent from these competitive binding assays, estetrol doesnot bind at all to human SHBG when tested with either [³H]DHT or[³H]estradiol as labeled ligands. This is in marked contrast withreference steroids ethinylestradiol, 17β-estradiol, testosterone and5α-dihydrotestosterone, which, in this order, show an increased relativebinding affinity for human SHBG. Importantly, estetrol binding to SHBGwas negligible when compared with the other estrogens tested,ethinylestradiol and 17β-estradiol.

Example 6

The present estrogenic components may suitably be processed, togetherwith additives, excipients and/or flavouring agents customary in galenicpharmacy, in accordance with the conventional methods into the usualforms of administration. For oral administration, suitable are, inparticular, tablets, dragees, capsules, pills, suspensions, orsolutions.

Estetrol tablets: 1,000 tablets of 185 mg, containing 1.5 mg estetroland 0.15 mg levonorgestrel, are produced from the following formulation:

Estetrol 1.500 g Levonorgestrel 0.150 g Polyvinylpyrrolidone 13.500 g(Kollidon 25® ex BASF) Lactose 135.645 g Microcrystalline cellulose(Avicel PH 101®) 26.250 g Glyceryl palmitostearate (Precirol ®) 2.775 gAnhydrous colloidal silica (Aerosil 200®) 1.000 g Crospovidone(Polyplasdone XL® ) 4.000 g Coloring agent 0.180 gTablets that additionally contain 50 mg dehydroepiandrosterone may beprepared from a similar formulation.

1. A method of hormone replacement in mammals comprising orallyadministering an estrogenic component and a progestogenic component to amammal in an effective amount to treat symptoms of hypoestrogenism, theestrogenic component being selected from the group consisting ofestetrol; precursors capable of liberating estetrol when used in thepresent method, which precursors are derivatives of estetrol, whereinthe hydrogen atom of at least one of the hydroxyl groups has beensubstituted by an acyl radical of a hydrocarbon carboxylic, sulfonic orsulfamic acid of 1-25 carbon atoms; tetrahydrofuranyl;tetrahydropyranal; or a straight or branched chain glycosidic residuecontaining 1-20 glycosidic units per residue; and mixtures of estetroland the precursors.
 2. The method according to claim 1, wherein thesymptoms of hypoestrogenism are selected from the group consisting ofosteoporosis, arteriosclerosis, climacteric symptoms, cognitivedisturbances and Alzheimer's disease.
 3. The method according to claim1, wherein the method comprises the daily oral administration of theestrogenic component during a period of at least 10 days.
 4. The methodaccording to claim 3, wherein the method comprises the daily oraladministration, during a period of at least 10 days, of a combination ofthe estrogenic component and a progestogenic component.
 5. The methodaccording to claim 4, wherein the method comprises the daily oraladministration of the combination of the estrogenic component and theprogestogenic component during a period of at least 28 days.
 6. Themethod according to claim 4, wherein the method further comprises aninterval of at least 2 days during which no progestogenic component andno estrogenic component is administered and wherein the resultingdecrease in serum concentration of the progestogenic component and theestrogenic component induces menses.
 7. The method according to claim 4,wherein the method comprises the daily oral administration of theestrogenic component during a period of at least 28 days and wherein,following the combined administration of the estrogenic component andthe progestogenic component, the estrogenic component and noprogestogenic component are administered during 3-18 consecutive daysand the resulting decrease in serum concentration of the progestogeniccomponent induces menses.
 8. The method according to claim 1, whereinthe method comprises the at least once daily oral administration of theestrogenic component and the progestogenic component during a period ofat least 10 days.
 9. The method according to claim 1, wherein theestrogenic component is orally administered in an amount of less than 1mg per kg of bodyweight per day.
 10. The method according to claim 1,wherein the estrogenic component is orally administered in an amount ofat least 1 μg per kg of bodyweight per day.
 11. The method according toclaim 1, wherein the progestogenic component is administered in anamount which is equivalent to a daily oral dosage of 0.3 to 20 μglevonorgestrel per kg of bodyweight.
 12. A pharmaceutical kit comprisingat least 20 oral dosage units that contain the estrogenic component asdefined in claim 1 and/or the progestogenic component as defined inclaim 1 and/or an androgenic component, wherein at least 10 unitscontain between 0.01 and 20 mg of the estrogenic component, at least 10units contain the progestogenic component in an amount equivalent to30-750 μg levonorgestrel and at least 10 dosage units contain theandrogenic component in an amount equivalent to 5-250 mgdehydroepiandrosterone.
 13. The pharmaceutical kit according to claim12, comprising at least 10 oral dosage units that contain between 0.01and 20 mg of the estrogenic component, the progestogenic component in anamount equivalent to 30-750 μg levonorgestrel and the androgeniccomponent in an amount equivalent to 5-250 mg dehydroepiandrosterone.14. The pharmaceutical kit according to claim 12, wherein the androgeniccomponent is selected from the group consisting ofdehydroepiandrosterone (DHEA), danazol, gestrinone, testosterone esters,precursors capable of liberating these androgens when used in thepresent method and mixtures thereof.